Reproductive Hormones During Minipuberty Suggest Subtle Testicular Impairments in Boys With Hypospadias

113 Danish infants with hypospadias showed reduced Sertoli and Leydig cell markers during minipuberty, regardless of defect severity

Journal: The Journal of Clinical Endocrinology and Metabolism | Published: 2026-04-22 | Type: Prospective Cohort Study | PMID: 41229307 Authors: Leunbach TL et al., Aarhus University Hospital and Copenhagen University Hospital–Rigshospitalet, Denmark Funding/COI: AP Fund, Dagmar Marshalls Fund (charitable); no COI declared

Summary

Boys presenting with hypospadias at a Danish tertiary center showed consistently lower concentrations of testicular Sertoli cell markers (AMH and inhibin B) and the Leydig cell peptide INSL3 during the minipuberty window — the brief postnatal surge in reproductive hormones that offers a rare, non-invasive view into gonadal function. Paradoxically, FSH and testosterone were elevated rather than suppressed. Severity of the hypospadias did not predict the magnitude of hormonal deviation, suggesting the testicular signal is decoupled from the urethral phenotype.

Claims

• Median AMH was −0.23 SDS below the healthy Danish reference (P < .001); inhibin B was −0.38 SDS below (P < .001); INSL3 was −0.31 SDS below (P < .001) — all three are direct markers of testicular cell output
• FSH was +0.83 SDS above reference (P < .001); total testosterone +0.71 SDS (P < .001); free testosterone +0.83 SDS (P < .001)
• Androstenedione (−0.52 SDS) and DHEAS (−0.50 SDS) were also significantly lower
• LH, SHBG, 17-hydroxyprogesterone, and DHT did not differ significantly from reference
• 89 of 113 boys (79%) had distal hypospadias; proximal cases had lower birth weight centiles (P = .014) and placental weight centiles (P = .038) than distal cases
• Hormone SDS values did not differ significantly between distal and proximal hypospadias subgroups

Study Quality

This is a well-executed prospective cohort with a comprehensive hormone panel assessed during the narrow minipuberty window — a methodologically sound choice because minipuberty is the only period in early childhood when these reproductive hormones are measurably active without invasive stimulation. Converting raw concentrations to SDS against a population-matched Danish reference cohort is appropriate and strengthens comparability.

The critical limitation is the absence of a concurrent control group; the study relies on external reference standards, which introduces cohort-effect risk even if the reference population is well-matched. With 113 boys enrolled from 139 presenting (81% consent rate), selection bias is modest but present. Subgroup analyses by hypospadias grade are underpowered given the small proximal group, and the finding that hormonal deviation does not track with severity weakens any mechanistic claim tying the two together.

Red Flags

• No concurrent healthy comparison group — all comparisons are against a historical reference standard
• Single tertiary center creates referral bias; the case mix may not represent the full population of boys with hypospadias
• The paradoxical elevation of FSH and testosterone alongside reduced Sertoli/Leydig markers is not fully explained, and the authors do not attempt a mechanistic reconciliation
• Clinical significance is undefined: the magnitude of SDS deviations is small, and no follow-up data exist on pubertal or adult reproductive outcomes
• Funding source (charitable) and undisclosed COI status leave some uncertainty, though there is no obvious industry conflict here

Strengths

• Prospective design with standardized hormone sampling timed to minipuberty
• Broad panel covering both Sertoli cell (AMH, inhibin B) and Leydig cell (INSL3, testosterone, androstenedione) markers — triangulating rather than relying on a single biomarker
• Population-matched Danish reference standards rather than generic international norms
• Includes placental weight, an underused proxy for fetal growth and endocrine environment
• Adequate enrollment rate (81%) and median age at sampling (0.28 years) is appropriate for the biological window

Verdict

This paper makes a focused, well-timed measurement and finds a genuine signal: boys with hypospadias have subtly impaired testicular cell output during minipuberty, independent of how severe their urethral defect is. That finding is worth paying attention to because it raises a legitimate question about long-term reproductive health that the field has not resolved. What the paper cannot do — and does not claim to do — is tell us whether this hormonal fingerprint predicts anything downstream. It is solid hypothesis-generating work from a credible group, not a practice-changing result.